Measuring Retrograde Actin Flow in Neuronal Growth Cones.

Actin flow refers to the motion of the F-actin cytoskeleton and has been observed in many different cell types, especially in motile cells including neuronal growth cones. The direction of the actin flow is generally retrograde from the periphery toward the center of the cell. Actin flow can be harnessed for forward movement of the cell through substrate-cytoskeletal coupling; thus, a key function of actin flow is in cell locomotion. In this chapter, we illustrate three different methods of quantifying retrograde F-actin flow in growth cones derived from cultured Aplysia bag cell neurons. These methods include tracking the movement of surface marker beads as well as kymograph analysis of time-lapse sequences acquired by differential interference contrast (DIC) imaging or fluorescent speckle microscopy (FSM). Due to their large size, Aplysia neuronal growth cones are uniquely suited for these methods; however, they can also be applied to any other growth cones with clear F-actin-rich peripheral domains.

Posterior approach to correct for focal plane offsets in lattice light-sheet structured illumination microscopy.

Significance: Lattice light-sheet structured illumination microscopy (latticeSIM) has proven highly effective in producing three-dimensional images with super resolution rapidly and with minimal photobleaching. However, due to the use of two separate objectives, sample-induced aberrations can result in an offset between the planes of excitation and detection, causing artifacts in the reconstructed images. Aim: We introduce a posterior approach to detect and correct the axial offset between the excitation and detection focal planes in latticeSIM and provide a method to minimize artifacts in the reconstructed images. Approach: We utilized the residual phase information within the overlap regions of the laterally shifted structured illumination microscopy information components in frequency space to retrieve the axial offset between the excitation and the detection focal planes in latticeSIM. Results: We validated our technique through simulations and experiments, encompassing a range of samples from fluorescent beads to subcellular structures of adherent cells. We also show that using transfer functions with the same axial offset as the one present during data acquisition results in reconstructed images with minimal artifacts and salvages otherwise unusable data. Conclusion: We envision that our method will be a valuable addition to restore image quality in latticeSIM datasets even for those acquired under non-ideal experimental conditions.

Dynamic Live Cell Imaging of Budding Yeast Meiosis.

For over a century, major advances in understanding meiosis have come from the use of microscopy-based methods. Studies using the budding yeast, Saccharomyces cerevisiae, have made important contributions to our understanding of meiosis because of the facility with which budding yeast can be manipulated as a genetic model organism. In contrast, imaging-based approaches with budding yeast have been constrained by the small size of its chromosomes. The advent of advances in fluorescent chromosome tagging techniques has made it possible to use yeast more effectively for imaging-based approaches as well. This protocol describes live cell imaging methods that can be used to monitor chromosome movements throughout meiosis in living yeast cells.

Computational Tools for the Analysis of Meiotic Prophase I Images.

Prophase I is a remarkable stage of meiotic division during which homologous chromosomes pair together and exchange DNA by meiotic recombination. Fluorescence microscopy of meiotic chromosome spreads is a central tool in the study of this process, with chromosome axis proteins being visualized as extended filaments upon which recombination proteins localize in focal patterns.Chromosome pairing and recombination are dynamic processes, and hundreds of recombination foci can be present in some meiotic nuclei. As meiotic nuclei can exhibit significant variations in staining patterns within and between nuclei, particularly in mutants, manual analysis of images presents challenges for consistency, documentation, and reproducibility. Here we share a combination of complementary computational tools that can be used to partially automate the quantitative analysis of meiotic images. (1) The segmentation of axial and focal staining patterns to automatically measure chromosome axis length and count axis-associated (and non-axis associated) recombination foci; (2) Quantification of focus position along chromosome axes to investigate spatial regulation; (3) Simulation of random distributions of foci within the nucleus or along the chromosome axes to statistically investigate observed foci-axis associations and foci-foci associations; (4) Quantification of chromosome axis proximity to investigate relationships with chromosome synapsis/asynapsis; (5) Quantification of and orientation of focus-axis distances. Together, these tools provide a framework to perform routine documentation and analysis of meiotic images, as well as opening up routes to build on this initial output and perform more detailed analyses.

Confocal Microscopy of Fluorescently Labeled EHDV-Infected Cell Cultures.

The confocal laser scanning microscope allows the visualization of intracellular structures in greater detail than a widefield fluorescence microscope. Immunofluorescence (IF) techniques make use of the inherent ability of antibodies to bind to specific epitopes of specific proteins. Tagging these antibodies with an easily visualized molecule, e.g., a fluorophore, enables imaging in the fluorescence microscope. This is, however, a localization technique and will only give information about where certain proteins are; it does not provide the ultrastructural context provided by the transmission electron microscope. It also relies heavily on the accuracy and binding affinity of individual primary antibodies. Despite this, it is a commonly used, robust, and adaptable technique. In this chapter, we use a long-established IF protocol from our laboratory to locate EHDV proteins in a monolayer of infected cultured cells.

Autofluorescence-based tissue characterization enhances clinical prospects of light-sheet-microscopy.

Light sheet fluorescence microscopy (LSFM) is a transformative imaging method that enables the visualization of non-dissected specimen in real-time 3D. Optical clearing of tissues is essential for LSFM, typically employing toxic solvents. Here, we test the applicability of a non-hazardous alternative, ethyl cinnamate (ECi). We comprehensively characterized autofluorescence (AF) spectra in diverse murine tissues-ocular globe, knee, and liver-employing LSFM under various excitation wavelengths (405-785 nm) to test the feasibility of unstained samples for diagnostic purposes, in particular regarding percutaneous biopsies, as they constitute to most harvested type of tissue sample in clinical routine. Ocular globe structures were best discerned with 640 nm excitation. Knee tissue showed complex variation in AF spectra variation influenced by tissue depth and structure. Liver exhibited a unique AF pattern, likely linked to vasculature. Hepatic tissue samples were used to demonstrate the compatibility of our protocol for antibody staining. Furthermore, we employed machine learning to augment raw images and segment liver structures based on AF spectra. Radiologists rated representative samples transferred to the clinical assessment software. Learning-generated images scored highest in quality. Additionally, we investigated an actual murine biopsy. Our study pioneers the application of AF spectra for tissue characterization and diagnostic potential of optically cleared unstained percutaneous biopsies, contributing to the clinical translation of LSFM.

A Fluorogenic Pseudoinfection Assay to Probe Transfer and Distribution of Influenza Viral Contents to Target Vesicles.

Fusion of enveloped viruses with endosomal membranes and subsequent release of the viral genome into the cytoplasm are crucial to the viral infection cycle. It is often modeled by performing fusion between virus particles and target lipid vesicles. We utilized fluorescence microscopy to characterize the kinetic aspects of the transfer of influenza viral ribonucleoprotein (vRNP) complexes to target vesicles and their spatial distribution within the fused volumes to gain deeper insight into the mechanistic aspects of endosomal escape. The fluorogenic RNA-binding dye QuantiFluor (Promega) was found to be well-suited for direct and sensitive microscopic observation of vRNPs which facilitated background-free detection and kinetic analysis of fusion events on a single particle level. To determine the extent to which the viral contents are transferred to the target vesicles through the fusion pore, we carried out virus-vesicle fusion in a side-by-side fashion. Measurement of the Euclidean distances between the centroids of superlocalized membrane and content dye signals within the fused volumes allowed determination of any symmetry (or the lack thereof) between them as expected in the event of transfer (or the lack thereof) of vRNPs, respectively. We found that, in the case of fusion between viruses and 100 nm target vesicles, ∼39% of the events led to transfer of viral contents to the target vesicles. This methodology provides a rapid, generic, and cell-free way to assess the inhibitory effects of antiviral drugs and therapeutics on the endosomal escape behavior of enveloped viruses.

ß-Galactosidase- and Photo-Activatable Fluorescent Probes for Protein Labeling and Super-Resolution STED Microscopy in Living Cells.

We report on the synthesis of two fluorescent probes which can be activated by ß-Galactosidase (ß-Gal) enzymes and/or light. The probes contained 2-nitro-4-oxybenzyl and 3-nitro-4-oxybenzyl fragments, with ß-Gal residues linked to C-4. We performed the enzymatic and photoactivation of the probes in a cuvette and compared them, prior to the labeling of Vimentin-Halo fusion protein in live cells with overexpressed ß-galactosidase. The dye fluorescence afforded the observation of enzyme activity by means of confocal and super-resolution optical microscopy based on stimulated emission depletion (STED). The tracing of enzymatic activity with the retention of activated fluorescent products inside cells was combined with super-resolution imaging as a tool for use in biomedicine and life science.

Placental Element Content Assessed via Synchrotron-Based X-ray Fluorescence Microscopy Identifies Low Molybdenum Concentrations in Foetal Growth Restriction, Postdate Delivery and Stillbirth.

Placental health and foetal development are dependent upon element homeostasis. Analytical techniques such as mass spectroscopy can provide quantitative data on element concentrations in placental tissue but do not show spatial distribution or co-localisation of elements that may affect placental function. The present study used synchrotron-based X-ray fluorescence microscopy to elucidate element content and distribution in healthy and pathological placental tissue. The X-ray fluorescence microscopy (XFM) beamline at the Australian Synchrotron was used to image trace metal content of 19 placental sections from healthy term (n = 5, 37-39 weeks), foetal growth-restricted (n = 3, <32 weeks, birth weight <3rd centile), postdate (n = 7, >41 completed weeks), and stillbirth-complicated pregnancies (n = 4, 37-40 weeks). Samples were cryo-sectioned and freeze-dried. The concentration and distribution of fourteen elements were detected in all samples: arsenic, bromine, calcium, chlorine, copper, iron, molybdenum, phosphorous, potassium, rubidium, selenium, strontium, sulphur, and zinc. The elements zinc, calcium, phosphorous, and strontium were significantly increased in stillbirth placental tissue in comparison to healthy-term controls. Strontium, zinc, and calcium were found to co-localise in stillbirth tissue samples, and calcium and strontium concentrations were correlated in all placental groups. Molybdenum was significantly decreased in stillbirth, foetal growth-restricted, and postdate placental tissue in comparison to healthy-term samples (p < 0.0001). Synchrotron-based XFM reveals elemental distribution within biological samples such as the placenta, allowing for the co-localisation of metal deposits that may have a pathological role. Our pilot study further indicates low concentrations of placental molybdenum in pregnancies complicated by foetal growth restriction, postdate delivery, and stillbirth.

Label free tracking to quantify nanoparticle diffusion through biological media.

Nanotechnology is a rapidly evolving field and has been extensively studied in biological applications. An understanding of the factors that influence nanoparticle diffusion in biofluids can aid in the development of diverse technologies. The development of real-time, label-free tracking technologies would allow the expansion of current knowledge of the diffusion and activity of nanoparticles. Fluorescence-based microscopy is one of the most widespread tools to monitor and track nanoparticle dynamics; however, the influence of fluorescent tags on diffusion and biological activity is still unclear. In this study, we experimentally determined the diffusion coefficient of gold nanoparticles using a label-free, optical tracking technique and evaluated the influence of protein concentration, charge and diameter on nanoparticle diffusion through biological media. We dispersed positively- and negatively-charged nanoparticles with diameters varying from 10 to 100 nm in a common cell culture media with different concentrations of serum proteins. Our results show that dynamic protein interactions influence nanoparticle diffusion in the range of serum concentrations tested. Experimental regimes to obtain quantitative information on the factors that influence the dynamics of nanoparticles in biological media have been developed.

Nanoscale single-vesicle analysis: High-throughput approaches through AI-enhanced super-resolution image analysis.

The analysis of membrane vesicles at the nanoscale level is crucial for advancing the understanding of intercellular communication and its implications for health and disease. Despite their significance, the nanoscale analysis of vesicles at the single particle level faces challenges owing to their small size and the complexity of biological fluids. This new vesicle analysis tool leverages the single-molecule sensitivity of super-resolution microscopy (SRM) and the high-throughput analysis capability of deep-learning algorithms. By comparing classical clustering methods (k-means, DBSCAN, and SR-Tesseler) with deep-learning-based approaches (YOLO, DETR, Deformable DETR, and Faster R-CNN) for the analysis of super-resolution fluorescence images of exosomes, we identified the deep-learning algorithm, Deformable DETR, as the most effective. It showed superior accuracy and a reduced processing time for detecting individual vesicles from SRM images. Our findings demonstrate that image-based deep-learning-enhanced methods from SRM images significantly outperform traditional coordinate-based clustering techniques in identifying individual vesicles and resolving the challenges related to misidentification and computational demands. Moreover, the application of the combined Deformable DETR and ConvNeXt-S algorithms to differently labeled exosomes revealed its capability to differentiate between them, indicating its potential to dissect the heterogeneity of vesicle populations. This breakthrough in vesicle analysis suggests a paradigm shift towards the integration of AI into super-resolution imaging, which is promising for unlocking new frontiers in vesicle biology, disease diagnostics, and the development of vesicle-based therapeutics.

Flagella and Cell Body Staining of Bacteria with Fluorescent Dyes.

Bacteria can propel themselves by rotating a flagellum or a flagellar bundle. To image this thin structure in motile bacteria, the flagella can be vitally stained with fluorophores. This chapter describes a flagellar staining protocol with the additional possibility of visualizing the cell body. It offers the opportunity to track conformational changes of flagella and simultaneously track the positions of the cell bodies. The additional use of a filter increases the number of motile cells and improves the signal-to-noise ratio of images. The flagellar staining requires a prior introduction of a surface-exposed cysteine, which is not covered in this chapter.

Fluorescence Lifetime Multiplexing (FLEX) for simultaneous high dimensional spatial biology in 3D.

Immunohistochemistry is a crucial method for detecting specific proteins within tissue samples, yet constrained to one biomarker per tissue section. Multiplexed immunofluorescence, while allowing simultaneous visualization of multiple proteins, faces limitations in the number of simultaneous fluorescent labels due to spectral overlap. Although cyclic immunofluorescence techniques have successfully broadened antibody staining capacities in a single tissue sample, they are plagued by time-consuming and labor-intensive procedures, sample degradation risks, and inability to scale beyond thin sections. In this study, we introduce the use of 3D confocal Fluorescence Lifetime Imaging Microscopy as a high-throughput, multiplexed immunofluorescence platform that can differentiate 11 or more biomarkers in 3D tissue volumes. Leveraging both spectral and lifetime information, this approach allows for practical spatial biology in thin sections that can readily scale to larger volumes of tissue. We believe that this highly multiplexed and versatile biomarker imaging platform will significantly expedite cancer research and enable new translational approaches in the future.

Flexible solid immersion meniscus lens (SIMlens) approach for enhancing biological imaging of cleared samples.

Tissue-clearing techniques have revolutionized the field of biological imaging by rendering biological specimens transparent and enabling inside optical detection. Light-sheet fluorescence microscopy (LSFM) is a powerful tool for three-dimensional imaging of large biological samples. Combining tissue-clearing techniques with LSFM has advanced the efficient 3D visualization of these samples. A crucial challenge with LSFM is the requirement for the objective to operate within the clearing reagent, which can cause aberrations. To address this issue, we introduce a novel, to our knowledge, approach for the flexible design of the solid immersion refractive meniscus lens (SIMlens), facilitating the use of air objectives with cleared samples. Compared to the previous SIMlens, this method not only eliminates aberrations but also offers customized options for enhancing the numerical aperture and working distance of the objective lens, achieving at least a 10% improvement. We have demonstrated the feasibility of this new method using mouse brain samples.

Fluorescence Microscopy and Immunoblotting for Mitophagy in Budding Yeast.

Selective removal of excess or damaged mitochondria is an evolutionarily conserved process that contributes to mitochondrial quality and quantity control. This catabolic event relies on autophagy, a membrane trafficking system that sequesters cytoplasmic constituents into double membrane-bound autophagosomes and delivers them to lysosomes (vacuoles in yeast) for hydrolytic degradation and is thus termed mitophagy. Dysregulation of mitophagy is associated with various diseases, highlighting its physiological relevance. In budding yeast, the pro-mitophagic single-pass membrane protein Atg32 is upregulated under prolonged respiration or nutrient starvation, anchored on the surface of mitochondria, and activated to recruit the autophagy machinery for the formation of autophagosomes surrounding mitochondria. In this chapter, we provide protocols to assess Atg32-mediated mitophagy using fluorescence microscopy and immunoblotting.

Analysis of Mitophagy Reporters in Drosophila.

Mitophagy is the degradation of mitochondria via the autophagy-lysosome system, disruption of which has been linked to multiple neurodegenerative diseases. As a flux process involving the identification, tagging, and degradation of subcellular components, the analysis of mitophagy benefits from the microscopy analysis of fluorescent reporters. Studying the pathogenic mechanisms of disease also benefits from analysis in animal models in order to capture the complex interplay of molecular and cell biological phenomena. Here, we describe protocols to analyze mitophagy reporters in Drosophila by light microscopy.

Quantitative Super-Resolution Imaging of ER-Phagy Initiation in Cells.

Selective autophagy of the endoplasmic reticulum (ER-phagy) is a mechanism that is necessary for degrading damaged ER components and preventing cells from experiencing ER stress. Various ER-phagy receptors orchestrate this process by building protein assemblies with dedicated functions. In order to understand the molecular building principles of ER-phagy, it is important to reveal the assembly of ER-phagy receptors in a temporal and functional context. However, direct visualization is hampered by the diffraction limit in light microscopy. Super-resolution microscopy (SRM) can bypass this limitation and resolve single proteins and nanoscale protein clusters in cells. In particular, DNA points accumulation for imaging in nanoscale topography (DNA-PAINT) is a powerful technology that can resolve individual protein clusters in cells and provide information on their molecular composition. Here, we report a step-by-step protocol on how to utilize DNA-PAINT to perform super-resolution imaging of ER-phagy receptors in fixed cells. In addition, we provide a detailed explanation of image generation, cluster analysis, and molecular quantification.

Fluorescence Methods to Measure Pexophagy.

We outline our approach for studying the selective autophagy of peroxisomes (pexophagy), using fluorescence microscopy in tissue cell culture models. Ratiometric reporters, which specifically localize to peroxisomes, allow a quantitative assessment of pexophagy in fixed and live cells, as well as whole organisms. We discuss chemical and physiological inducers of pexophagy and any overlap with the induction of mitophagy.

Making Rift Valley Fever Viral Particles Fluorescent.

Rift Valley fever virus (RVFV) is a mosquito-borne pathogen that represents a significant threat to both human and veterinary public health. Since its discovery in the Great Rift Valley of Kenya in the 1930s, the virus has spread across Africa and beyond, now posing a risk of introduction into Southern Europe and Asia. Despite recent progresses, early RVFV-host cell interactions remain largely uncharacterized. In this method chapter, we delineate the procedure for labeling RVFV particles with fluorescent organic dyes. This approach makes it feasible to visualize single viral particles in both fixed and living cells and study RVFV entry into host cells. We provide additional examples with two viruses closely related to RVFV, namely, Toscana virus and Uukuniemi virus. Furthermore, we illustrate how to utilize fluorescent viral particles to examine and quantify each step of the cell entry program of RVFV, which includes state-of-the-art fluorescence-based detection techniques such as fluorescence microscopy, flow cytometry, and fluorimetry.

Self-Quenched Fluorophore-DNA Labels for Super-Resolution Fluorescence Microscopy.

Protein labeling through transient and repetitive hybridization of short, fluorophore-labeled DNA oligonucleotides has become widely applied in various optical super-resolution microscopy methods. The main advantages are multitarget imaging and molecular quantification. A challenge is the high background signal originating from the presence of unbound fluorophore-DNA labels in solution. Here, we report the self-quenching of fluorophore dimers conjugated to DNA oligonucleotides as a general concept to reduce the fluorescence background. Upon hybridization, the fluorescence signals of both fluorophores are restored. We expand the toolbox of fluorophores suitable for self-quenching and report their spectra and hybridization equilibria. We apply self-quenched fluorophore-DNA labels to stimulated emission depletion microscopy and single-molecule localization microscopy and report improved imaging performances.